Discovery and Characterization of Brigimadlin, a Novel and Highly Potent MDM2-p53 Antagonist Suitable for Intermittent Dose Schedules

Abstract

p53 is known as the guardian of the genome and is one of the most important tumor suppressors. It is inactivated in most tumors, either via tumor protein p53 (TP53) gene mutation or copy number amplification of key negative regulators, e.g., mouse double minute 2 (MDM2). Compounds that bind to the MDM2 protein and disrupt its interaction with p53 restore p53 tumor suppressor activity, thereby promoting cell cycle arrest and apoptosis. Previous clinical experience with MDM2-p53 protein-protein interaction antagonists (MDM2-p53 antagonists) has demonstrated that thrombocytopenia and neutropenia represent on-target dose-limiting toxicities that might restrict their therapeutic utility. Dosing less frequently, while maintaining efficacious exposure, represents an approach to mitigate toxicity and improve the therapeutic window of MDM2-p53 antagonists. However, to achieve this, a molecule possessing excellent potency and ideal pharmacokinetic properties is required. Here, we present the discovery and characterization of brigimadlin (BI 907828), a novel, investigational spiro-oxindole MDM2-p53 antagonist. Brigimadlin exhibited high bioavailability and exposure, as well as dose-linear pharmacokinetics in preclinical models. Brigimadlin treatment restored p53 activity and led to apoptosis induction in preclinical models of TP53 wild-type, MDM2-amplified cancer. Oral administration of brigimadlin in an intermittent dosing schedule induced potent tumor growth inhibition in several TP53 wild-type, MDM2-amplified xenograft models. Exploratory clinical pharmacokinetic studies (NCT03449381) showed high systemic exposure and a long plasma elimination half-life in patients with cancer who received oral brigimadlin. These findings support the continued clinical evaluation of brigimadlin in patients with MDM2-amplified cancers, such as dedifferentiated liposarcoma.

Graphical abstract

Figure 1.

Discovery and structure of brigimadlin. A, Development of brigimadlin. B, X-ray co-crystal structure of brigimadlin. CTG, CellTiter-Glo.

Figure 2.

Sensitivity to brigimadlin in the PRISM screen assay. A, Sensitivity to brigimadlin in 683 cell lines with a nonzero activity area and color coded by TP53 mutation status. Cell lines are arranged from left to right in order of decreasing sensitivity to brigimadlin. The green (top) and purple (bottom) horizontal dashed bars reflect the cutoffs used for sensitive and nonsensitive (resistant) cell lines, respectively. B, Sensitivity to brigimadlin in 266 TP53 wild-type cell lines grouped by disease type. The green and purple dashed bars reflect the cutoffs used for sensitive and nonsensitive (resistant) cell lines, respectively. The short red marks highlight the respective groups’ median. Note that only types of cancers with ≥3 cell lines are shown on the plot. mut, mutated; wt, wild type; * tumor type not defined.

Figure 3.

MDM2 expression and brigimadlin dose-dependent regulation of p53 target genes and apoptosis in MDM2-amplified and non-amplified solid tumor cell lines. A, MDM2 mRNA (Ai) and protein (Aii) expression in MDM2-amplified and non-amplified cell lines. B, Brigimadlin dose-dependent upregulation of mRNA of seven genes in MDM2-amplified (SJSA-1) and non-amplified (U-2 OS) solid tumor cell lines (mean of technical replicates ± standard deviation). Bi, PUMA (BBC3); Bii, P21 (CDKN1A); Biii, MDM2; Biv, CD80; Bv, BAX; Bvi, BIM; Bvii, BCL-XL. One-step RT-PCR normalized to GAPDH: PUMA and P21 (24 hours) and BAX, BIM, and BCL-XL (48 hours). Two-step RT-PCR normalized to GAPDH: MDM2 and CD80 (48 hours). C, Western blot of biomarker proteins in SJSA-1 and U-2 OS cell lines after 24 and 72-hour treatment with brigimadlin. D, Brigimadlin dose and time dependence of cleaved PARP (Di) and cleaved caspase-3 (Dii) signal relative to vehicle (MSD apoptosis assay). amp, amplified; cl, cleaved; h, hour; rel, relative; TPM, transcripts per million.

Figure 4.

Modulation of p53 target genes and markers of apoptosis in SJSA-1 tumor-bearing mice treated with brigimadlin. A, Mean (± SEM) change in GDF15 (MIC-1) levels in serum samples 24 hours after a single oral dose of brigimadlin. SJSA-1 tumor-bearing mice were treated with a single oral dose of either 0.25, 0.75, 2, or 10 mg/kg brigimadlin, respectively, or with vehicle. Absolute GDF15 levels are depicted in pg/mL serum. Error bars represent the SEM of three biological repeats. B, Mean (± SEM) change in mRNA levels of p53 target genes in SJSA-1 tumor samples 24 hours after a single oral dose of brigimadlin. SJSA-1 tumor-bearing mice were treated with a single oral dose of 0.25, 0.75, 2, or 10 mg/kg brigimadlin, respectively, or vehicle. Changes of mRNA levels are depicted as fold change relative to the vehicle control. Error bars represent the SEM of three biological repeats. C, Correlation of GDF15 protein levels from serum samples with fold change GDF15 mRNA in tumor samples. D, Brigimadlin concentration–time curve in plasma of SJSA-1 tumor-bearing mice treated with either brigimadlin 2 mg/kg single dose or 1 mg/kg BID × 1 day. Brigimadlin plasma concentration over time is shown. E, Dose-dependent mRNA modulation of p53 target genes in SJSA-1 tumors harvested at indicated posttreatment time points (two-step RT-PCR data normalized to GAPDH and relative to respective control): Ei, p21 (CDKN1A); Eii, MDM2; Eiii, GDF15; Eiv, PUMA (BBC3); Ev, CD80; Evi, BAX; Evii, BIM; Eviii, BCL-XL. F, Dose- and time-dependent modulation of apoptotic markers (Fi, cleaved PARP and Fii, cleaved caspase-3) in SJSA-1 tumors harvested at indicated posttreatment time points (MSD apoptosis assay). BID, twice daily; Cl, cleaved; d, day; h, hours; rel, relative; SD, standard deviation; SEM, standard error of the mean.

Figure 5.

Brigimadlin efficacy in mouse xenograft models. Tumor growth curves (mean ± SEM) for brigimadlin in A, MDM2-amplified SJSA-1 osteosarcoma CDX model [MDM2 CN (qPCR) = 55]; (B), MDM2-amplified undifferentiated pleomorphic sarcoma (SA3283) PDX model [MDM2 CN (qPCR) = 132]; (C), MDM2-amplified lung squamous cell carcinoma (LU0861) PDX model [MDM2 CN (qPCR) = 20]; (D), MDM2-amplified lung squamous cell carcinoma (LU6903) PDX model [MDM2 CN (qPCR) = 19]. Ten mice per treatment group were randomized to brigimadlin 2 mg/kg p.o. QW for 2 weeks (SJSA-1 and SA3283), 3 weeks (LU0861), or 5 weeks (LU6903)], respectively, or to vehicle. BID, twice daily; CDX, cell line-derived xenograft; CN, copy number; PDX, patient-derived xenograft; p.o., oral; QW, once per week; SEM, standard error of the mean. P-values (brigimadlin vs. vehicle control group) of <0.05 are regarded as statistically significant.